Figure 4: Lane 2 has 1kb DNA ladder, Lane 3 has uncut plasmid, Lane 4 has sample 1 with EcoRI, Lane 5 has sample 2 with PvuII, and Lane 6 has sample 3 with EcoRI + PvuII.
Image 5: (PCR1) Lane 2 has 100bp ladder, Lane 3 has 1 ul of 1:1000 dilution of pGBR22 (.3ng), Lane 4 has 10ul 1:1000 dilution of pGBR22 (3ng), Lane 5 has 10ul 1:100 dilution pGBR22 (30ng), and Lane 6 had no DNA.
Image 6:(PCR2) Lane 2 has 100bp ladder, Lane 3 had .008ng/ul pmCherry, Lane 4 had .08ng/ul pmCherry, Lane 5 had .8ng/ul pmCherry, Lane 6 had no DNA template; Lanes 3-6 had the forward primer VDSR1 and reverse primer VDSR2. Lane 7 had .008ng/ul pmCherry, Lane 8 had .08ng/ul pmCherry, Lane 9 had .8ng/ul pmCherry, Lane 10 had no DNA template; Lanes 7-10 had the primer M13 Forward and M13 Reverse. Lanes 11 and 12 were testing ladders.
Any thoughts on why the left side didn't work? - Dr. B
The anneal temperature during PCR may have been too high.
Image 7: (PCR 3) Lane 2 100bp ladder, Lane 3 100bp ladder, Lane 4 .001ng/ul pNIC-Bsa4, Lane 5 .01ng/ul pNIC-Bsa4, Lane 6 .1ng/ul pNIC-Bsa4, and Lane 7 No DNA.
None of the experimental lanes appeared.
Image 9: Lane 2 Ladder, Lane 3 Cell lysate before induction, Lane 4 Cell lysate after induction, Lane 5 Soluble fraction, Lane 6 Flow Through, Lane 7 Wash, Lane 8 Elution 1, Lane 9 Elution 2
Lane 8 Elution 1 has a light band of the protein, ideally it should have been darker.
Lane 9 Elution 2 had nothing as expected.
Image 10: Trial 1; Lane 2 Ladder, Christina: Lane 3 Primary PCR & Lane 4 Secondary PCR, Jeanette: Lane 5 Primary PCR & Lane 6 Secondary PCR, Sadhana: Lane 7 Primary PCR & Lane 8 Secondary PCR, Joshua: Lane 9 Primary PCR & Lane 10 Secondary PCR
This gel was ran to confirm that PCR worked and oligo mix was made correctly. My first PCR had a slight smear and the second didn't. The PCR will be made again.
Image 11: Lane 2 Ladder, Lane 3 Primary PCR, Lane 4 Secondary PCR
Primary PCR had a slight smear and the secondary PCR had a band. Success.
Jeanette's Research
Transform Efficiency Calculations:
Sample A: 48/1ng * 1000ng/1ug = 4.8e^4 transformants/ug plasmid
Sample B: 144/5ng * 1000ng/1ug = 2.88e^4 transformants/ug plasmid
Sample C: 368/25ng * 1000ng/1ug = 1.472e^4 transformants/ug plasmid
Any thoughts on why the left side didn't work? - Dr. B
The anneal temperature during PCR may have been too high.
None of the experimental lanes appeared.
Lane 8 Elution 1 has a light band of the protein, ideally it should have been darker.
Lane 9 Elution 2 had nothing as expected.
This gel was ran to confirm that PCR worked and oligo mix was made correctly. My first PCR had a slight smear and the second didn't. The PCR will be made again.
Primary PCR had a slight smear and the secondary PCR had a band. Success.